Compositions and methods for treatment of hematological malignancies

ABSTRACT

Use of a chimeric protein selected from the group consisting of CTLA4-FasL and CD40-FasL proteins for treatment of lymphoma and/or a multiple myeloma and/or a leukemia as described herein, and pharmaceutical compositions and methods of treatment thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/824,423, which entered the National Stage in the United States under35 U.S.C. § 371 on Jul. 28, 2013, from International Application No.PCT/IB2011/054260, International Filing Date Sep. 28, 2011, claiming thebenefit of U.S. Provisional Patent Application No. 61/387,073, filedSep. 28, 2010, all of which are hereby incorporated by reference.

This invention relates to compositions and methods for treatment ofhematological malignancies, and in particular, but not exclusively, tofusion proteins and compositions and methods of use thereof.

BACKGROUND OF THE INVENTION

There are two major arms to the immune system, supported by differenttypes of cells called B-lymphocytes and T-lymphocytes (B-cells andT-cells). B cells make antibodies when they encounter antigens and, inmost instances, these antibodies are protective. In autoimmune diseases,however, some of the antibodies react with the individual's tissues.When they deposit in tissue, they cause an inflammatory reaction andtissue damage. T-cells, like B-cells, are also activated when theyencounter an antigen. As T-cells develop they undergo a process called“thymic education.” During thymic education, more than 95% of theT-cells die. The T-cells that have had a T-cell receptor that canrecognize and react with the individuals's own tissues (self-antigens)are specifically eliminated. Some autoreactive T-cells escape theelimination process, however, and can initiate an immune response thatresults in autoimmune disease.

The modulation of T-cell activity remains a significant therapeutic goalin diseases with immunopathological T-cells. The fate of T lymphocytesfollowing T cell receptor (TCR) stimulation is guided by the integrationof costimulatory and inhibitory receptor inputs. Costimulatory ligandson antigen-presenting cells (APC) trigger cognate receptor molecules onT cells, with resultant enhancement of T cell proliferation, cytokinesecretion, and differentiation. In contrast, binding of inhibitoryligand molecules to cognate counter-receptors on T lymphocytesdiminishes effector functioning by inducing T cell unresponsiveness orprogrammed cell death (PCD) (also referred to as apoptosis).Costimulatory and inhibitory receptor pathway interactions are suggestedby experiments demonstrating increased inhibitor activity in thepresence of costimulator blockade.

Cytotoxic T lymphocyte-associated protein-4 (CTLA-4 (CD152)) is aninhibitory receptor molecule that is expressed on the surface ofactivated T lymphocytes. Following engagement with the B7-1 (CD80)and/or B7-2 (CD86) ligands resident on APC, the CTLA-4 counter-receptor,via associated SHP-2 phosphatase, inhibits T cell activation. Onactivated T cells, CTLA-4 exists as disulfide-linked homodimericglycoprotein complexes. A recombinant, soluble CTLA-4:immunoglobulin G(CTLA-4:Ig) chimeric protein demonstrates inhibitory function bycompetitively blocking CD80/CD86 molecule binding to the activating CD28acceptor on T cell surfaces. CTLA-4:Ig also exhibits immunosuppressiveactivity in animal models of graft rejection and autoimmune disease byblocking T cell costimulation through CD28. In addition, intracellular Tcell survival signaling through CD28 is antagonized by APC treatmentwith CTLA-4:Ig, which can increase susceptibility to Fas-dependent PCD.The action of CTLA-4, as well as CTLA-4:Ig fusion proteins, arediscussed in U.S. Pat. Nos. 5,885,776; 5,885,579; 5,851,795; and5,968,510.

Apoptosis (or PCD) is a distinct form of cell death which is essentialfor the regulation of cellular homeostasis. In the immune system, Fas(CD95) receptor and its ligand, FasL (CD95L), participate in variousprocesses involved in the induction of apoptosis, including immunecell-mediated cytotoxicity, and in the regulation of cellular immuneresponses. FasL is a member of the tumor necrosis factor superfamily andis expressed by a restricted subset of immune cells, includingmonocytes, NK cells, and activated B and T cells. On the cell surface,FasL is oriented as a type II membrane protein within trimericcomplexes. Metalloproteinase cleavage of membrane-associated FasLreleases soluble FasL (sFasL) trimers from the membrane. The FasLmolecule triggers Fas-dependent PCD.

The valency of a molecule or molecular complex can be increased byassociation with the cell surface. Different coding sequences ofrecombinant sFasL molecules affect macromolecular aggregation and, inturn, affect sFasL pro-apoptotic function. In particular, a naturallyprocessed sFasL molecule forms trimers and poorly induces apoptosis. Incontrast, a recombinant full-length extracellular domain sFasLpolypeptide forms higher order aggregates and displays highly potentapoptotic activity. Furthermore, complexes of sFasL produced byrecombinant expression in human 293 cells require cross-linking forlysis of Fas-sensitive cells.

U.S. Pat. No. 5,830,469 discloses monoclonal antibodies and bindingproteins that specifically bind to human Fas antigen; some of theantigens and antibodies are reported as stimulating T cellproliferation, inhibiting of anti-Fas CH-11 monoclonal antibody-mediatedlysis of cells, and blocking Fas ligand-mediated lysis of cells. Fas-Fcfusion proteins are also disclosed.

U.S. Pat. Nos. 5,242,687; 5,601,828; and 5,623,056 disclose variousfusion proteins containing a CD8 component that bind to a cell but donot mask a signal produced by the cell.

U.S. Pat. No. 5,359,046 discloses chimeric proteins comprised of anextracellular domain capable of binding to a ligand in a non-MHCrestricted manner, a transmembrane domain and a cytoplasmic domaincapable of activating a signaling pathway. Similar technology isdisclosed in U.S. Pat. No. 5,686,281.

SUMMARY OF THE INVENTION

The background art does not teach or suggest methods of treatment oflymphoma and multiple myeloma through the administration of chimericproteins which are useful for both blocking and signaling.

The present invention, in at least some embodiments, overcomes thesedrawbacks of the background art by providing methods of treatment oflymphoma and/or multiple myeloma through the administration of chimericproteins which are useful for both blocking and signaling.

In at least some embodiments, there is provided pharmaceuticalcompositions containing such chimeric proteins, adapted for treatment oflymphoma and/or multiple myeloma.

By “lymphoma” it is meant a malignant growth of B or T cells in thelymphatic system, optionally including Hodgkin's lymphoma ornon-Hodgkin's lymphoma (NHL).

According to at least some embodiments, the non-Hodgkin's Lymphoma is aselected from the group consisting of aggressive NHL, transformed NHL,indolent NHL, relapsed NHL, refractory NHL, low grade non-Hodgkin'sLymphoma, follicular lymphoma, large cell lymphoma, B-cell lymphoma,T-cell lymphoma, Mantle cell lymphoma, Burkitt's lymphoma. NK celllymphoma, diffuse large B-cell lymphoma, acute lymphoblastic lymphoma,and cutaneous T cell cancer, including mycosos fungoides/Sezry syndrome.

An “indolent” non-Hodgkin's Lymphoma is a classification that includesslow growing forms of lymphoma. They encompass what are called low gradeand some categories of intermediate grade NHL in the WorkingFormulation. Indolent NHLs are sometimes not responsive to conventionalcancer therapies such as chemotherapy and radiation therapy. IndolentNHL and other premalignant forms of NHL may also proceed to NHL. Withregard to premalignant or benign forms of the disease, optionally thecompositions and methods thereof may be applied for prevention, inaddition to or in place of treatment, for example optionally to halt theprogression of the disease to a malignant form of NHL.

A “transformed” non-Hodgkin's Lymphoma is a classification sometimesemployed to describe an indolent NHL which acquires an aggressive aspectand becomes more responsive to standard chemotherapies.

By “multiple myeloma” it is meant any type of B-cell malignancycharacterized by the accumulation of terminally differentiated B-cells(plasma cells) in the bone marrow.

According to at least some embodiments, the multiple myeloma is selectedfrom the group consisting of multiple myeloma cancers which producelight chains of kappa-type and/or light chains of lambda-type; and/oraggressive multiple myeloma, including primary plasma cell leukemia(PCL); and/or optionally including benign plasma cell disorders such asMGUS (monoclonal gammopathy of undetermined significance) and/orWaldenström's macroglobulinemia (WM, also known as lymphoplasmacyticlymphoma) which may proceed to multiple myeloma; and/or smolderingmultiple myeloma (SMM), and/or indolent multiple myeloma, premalignantforms of multiple myeloma which may also proceed to multiple myeloma;and/or primary amyloidosis. With regard to premalignant or benign formsof the disease, optionally the compositions and methods thereof may beapplied for prevention, in addition to or in place of treatment, forexample optionally to halt the progression of the disease to a malignantform of multiple myeloma.

According to at least some embodiments of the present invention, thereis provided a method of treatment of a leukemia selected from the groupconsisting of acute nonlymphocytic leukemia, chronic lymphocyticleukemia, acute granulocytic leukemia, chronic granulocytic leukemia,acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia,a leukocythemic leukemia, basophylic leukemia, blast cell leukemia,bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cellleukemia, Schilling's leukemia, stem cell leukemia, subleukemicleukemia, and undifferentiated cell leukemia.

In at least some embodiments, there is provided pharmaceuticalcompositions containing such chimeric proteins, adapted for treatment ofa leukemia selected from the above group.

According to at least some embodiments of the present invention, thechimeric protein is selected from the group consisting of CTLA4-FasL andCD40-FasL proteins.

U.S. Pat. No. 7,569,663 to Tykocinski, et al., issued on Aug. 4, 2009,which is hereby incorporated by reference as if fully set forth herein,describes chimeric proteins that function as both a blocking protein anda signaling protein. Such chimeric proteins include CTLA4-FasL andCD40-FasL proteins. Data was also provided in this patent thatdemonstrates the efficacy of the CTLA-4-FasL chimeric protein to inhibitthe polyclonal proliferation of human peripheral blood T cells tomitogenic anti-CD3 antibody. Methods of use and of production are alsodescribed.

As used herein the term “treatment” refers to care provided to relieveillness and refers to both a therapeutic treatment orprophylactic/preventative measures, wherein the objective is to preventor slow down (lessen) the targeted pathologic condition or disorder.Those in need of treatment include those already with the disorder aswell as those prone to have the disorder or those in whom the disorderis to be prevented. The term treatment as used herein refers also to“maintenance therapy”, which is a treatment that is given to keep apathologic condition or disorder from coming back after it hasdisappeared following the initial therapy.

The term “therapeutically effective amount” refers to an amount of agentaccording to the present invention that is effective to treat a diseaseor disorder in a mammal.

According to any of the above described embodiments of chimeric proteinsand/or pharmaceutical compositions thereof, alone or in combination withother therapeutics or drugs (for combination therapy), there isprovided, according to at least some embodiments of the presentinvention, compositions and methods of treatment therefore for treatmentof a hematological malignancy as described herein. Such one or moreadditional therapeutics or drugs could easily be selected by one ofordinary skill in the art.

As used herein the term “combination therapy” refers to the simultaneousor consecutive administration of two or more medications or types oftherapy to treat a single disease, preferably with a synergistic effect.In particular, the term refers to the use of any of the chimericproteins or pharmaceutical compositions according to at least someembodiments of the invention in combination with at least one additionalmedication or therapy. Thus, treatment of a disease using the agentsaccording to at least some embodiments of the present invention may becombined with therapies well known in the art that include, but are notlimited to, radiation therapy, antibody therapy, chemotherapy or surgeryor in combination therapy with other biological agents, conventionaldrugs, anti-cancer agents, immunosuppressants, cytotoxic drugs forcancer, chemotherapeutic agents.

According to at least some embodiments, treatment of Multiple Myelomausing the agents according to at least some embodiments of the presentinvention may be combined with an agent including but not limited toMelphalan, thalidomide (MPT), or combination Bortezomib (Velcade),melphalan, prednisone (VMP) or a combination of Lenalidomide pluslow-dose dexamethasone; and/or biophosphonates; chemotherapy (e.g.,alkylating agents, vincristine, doxorubicin); autologous stem celltransplantation; and corticosteroids (e.g., prednisone anddexamethasone).

According to at least some embodiments, treatment of leukemia using theagents according to at least some embodiments of the present inventionmay be combined with an agent including but not limited toalpha-interferon: interleukin-2; cytarabine and mitoxantrone; cytarabineand daunorubicin and 6-thioguanine; cyclophosphamide and2-chloro-2′-deoxyadenosine; VP-16 and cytarabine and idorubicin ormitoxantrone; fludarabine and cytarabine and .gamma.-CSF; chlorambucil;cyclophosphamide and vincristine and (prednisolone or prednisone) andoptionally doxorubicin; tyrosine kinase inhibitor; and antibody;glutamine; clofibric acid; all-trans retinoic acid; ginseng diyneanalog; KRN8602 (anthracycline drug); temozolomide and poly(ADP-ribose)polymerase inhibitors; lysofylline; cytosine arabinoside; chlythorax andelemental enteral diet enriched with medium-chain triglycerides;amifostine; and gilvusmycin.

According to at least some embodiments, treatment of lymphoma using theagents according to at least some embodiments of the present inventionmay be combined with an agent including but not limited to a vincaalkaloid, such as vincristine, vinblastine, vindesine, or vinorelbine;an anthracycline such as doxorubicin; combinations such as CHOP(vincristine, cyclophosphamide, doxorubicin and prednisone); and othersuitable alkaloids including, but not limited to, the podophyllins,podophyllotoxins, and derivatives thereof (e.g., etoposide, etoposidephosphate, teniposide, etc.), the camptothecins (e.g., irinotecan,topotecan, etc.) the taxanes (taxol, etc.), and derivatives thereof.

As used herein, the term “synergistic effect” or “synergism” refers to agreater effect seen with a combination of a plurality of therapeuticagents, including at least one therapeutic agent according to anyembodiment of the present invention, in which the therapeutic effect isgreater than the additive effects of the plurality of agents whenadministered singly. By “greater therapeutic effect”, it is meant agreater cancer effect and/or a reduction in one or more side effects.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats,horses, cows, chickens, amphibians, reptiles, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Expression of fusion protein counter-receptors on T and Bmalignant cell lines. CD40L-expressing (J-CD40L+) and non-expressing(JCD40L−) malignant T cell lines, as well as Raji and Daudi malignant Bcell lines, were immuno-stained with FITC-conjugated anti-CD95,anti-CD80, anti-CD86 or anti-CD40L mAb (in black), or with isotypecontrol Ab (white), and then analyzed by flow cytometry, as described inMethods.

FIG. 2. Susceptibility to J-CD40L+ and J-CD40L− cells to CTLA-4⋅FasL andCD40⋅FasL is dependent on expression of the relevant counterreceptors.A. CD40L-expressing (J-CD40L+) and non-expressing (J-CD40L−) malignant Tcells (upper two panels), and Raji and Daudi malignant B cells (lowertwo panels), were plated (0.5×105) in round-bottom, 96-well plates inthe presence or absence of CTLA-4⋅FasL, CD40⋅FasL, or sFas (30 ng/mleach), or CTLA-4⋅Ig or CD40-Fc (100 ng/ml each, or indicatedcombinations. Cells were pulsed with [3H]thymidine and incubated for 18h. Assays were performed in triplicate. Data is presented as percentageof [3H]thymidine incorporation of cells in incubated in growth medium.The results shown summarize 3 independent experiments for each cellline. *P<0.05 versus control, **P<0.01 versus control. B. 0.5×105 Rajicells were incubated with (black bars) or without (white bars)CTLA-4⋅FasL (30 ng/ml) in the presence of either anti-CD80 or anti-CD86Ab (B7 blockers) or their combination for 18 h. Proliferation wasdetermined and presented as in A. These results summarize threeindependent experiments. p<0.05, **p<0.01 vs. medium.

FIG. 3. Susceptibility of J-CD40L+ and J-CD40L− cells to apoptosisinduction by CTLA-4⋅FasL and CD40⋅FasL is dependent on their expressionof relevant counter-receptors. J-CD40L+, CD40L (J-CD40L−). Raji andDaudi cells were incubated in the presence or absence of eitherCTLA-4⋅FasL, CD40⋅FasL, or sFas (30 ng/ml each), or CTLA-4⋅Ig or CD40-Fc(100 ng/ml each, or indicated combinations, for 4 h (T cells) or 16 h (Bcells). 26 Cells were harvested, co-stained with Annexin V and PI, andanalyzed by flow cytometry. A. A representative dot plot analysis of thecell lines in the presence or in the absence of CTLA-4⋅FasL orCD40⋅FasL. B-C. Percentage of dead cells (Annexin V+/PI−+Annexin V+/PI+)obtained from FACS analysis of Jurkat malignant T cell lines (B), Rajiand Daudi malignant B cell lines (C). The summary of three independentexperiments is shown. Data are presented as mean±SD. **p<0.01 vs.medium.

FIG. 4. CTLA-4⋅FasL and CD40-FasL each affects both apoptotic andanti-apoptotic signaling pathway elements. J-CD40L+J-CD40L−, Raji andDaudi cells were incubated in the presence or absence of eitherCTLA-4⋅FasL, CD40⋅FasL, sFas, CTLA-4⋅Ig, CD40-Fc or their combinationsas indicated, for 90 min. Cells were collected, whole cell lysates werefractionated on 10% SDS-PAGE, and immunoblotted with the indicated Ab.A. Representative immunoblots for Jurkat malignant T cell lines and Rajiand Daudi malignant B cell lines. Data shown is a representativeexperiment of at least three independent experiments for each cell line.B-C. Summary of three independent experiments with the indicated Ab forRaji (B) and Jurkat (C) cell lines. The active forms of caspases 3, 9and 8 are shown. Data were normalized against GAPDH. Expression ofproteins in cells incubated in medium (control) were considered as 100%.Data are presented as mean±SD. *p<0.05 vs. medium, **p<0.01 vs. medium.

FIG. 5. CTLA4⋅FasL increases apoptotic signals and decrease theanti-apoptotic protein cFLIP in JY malignant B cells. JY cells wereincubated in the presence or absence of either CTLA-4⋅FasL, CD40⋅FasL oranti-Fas Ab (CH11) for 90 min. Cells were collected, whole cell lysateswere fractionated on 10% SDS-PAGE, and immunoblotted with the indicatedAb. A. Representative immunoblots of three independent experiments foreach cell line. B Summary of three independent experiments of JYmalignant B cell lines with cFLIPs and anti-caspase 8 Ab C Summary ofthree independent experiments of JY malignant B cell lines withanti-caspase 9 and 3 Ab. Expression of proteins in cells incubated inmedium (control) were considered as 100%. Data are presented as mean±SD.*p<0.05 vs. medium, **p<0.01 vs. medium.

FIG. 6. CTLA-4⋅Ig reduces proliferation of B7-expressing cells andaffects their expression of caspases and cFLIP. A. Raji malignant Bcells were incubated in the presence or absence of CTLA-4⋅FasL onflat-bottom 96-well plates that were pre-incubated with CTLA-4⋅Igovernight. Assays were performed in triplicate. Cells were pulsed with[3H]thymidine, incubated for 24 h and then evaluated for [3H]thymidineincorporation. [3H]thymidine incorporation of cells in medium (control)was designated as 100%, and the rest are presented as % of control,mean±SD. *P<0.05 versus control, **P<0.01 versus control. Data shown area summary of three independent experiments. B-C. Raji cells wereincubated on 24-well plates pre-coated with CTLA-4⋅Ig for 90 min. Cellswere collected, whole cell lysates were fractionated on 10% SDS-PAGE,and immunoblotted with the indicated Ab. B. Representative immunoblotswith the indicated Ab. C. Summary of three independent experiments. Theactive forms of caspases 3, 9 and 8 are shown. Data were normalizedagainst GAPDH. Expression of proteins in cells incubated in medium(control) were considered as 100%. Data are presented as mean±SD.*p<0.05 vs. medium.

FIGS. 7 and 8 show that CTLA4-FasL induces death of both Raji and JY,B-cell lymphatic cancer cell lines, in a dose-dependent fashion.

FIG. 9 shows the effect of CTLA4-FasL on RPMI 8226.

FIG. 10 shows the effect of CTLA4-FasL on promyelocytic leukemia cells.

FIGS. 11-14 relate to the surface expression of CD80 and CD86, whichbind CTAL4, and the expression of CD95 (Fas receptor, which binds FasL),in various cell lines.

FIG. 15 shows that CTLA4-FasL exhibited a cytotoxic effect againstSK-Hep1 hepatoma cells.

FIG. 16 shows that the pan-caspase inhibitor, zVAD, completely abolishedCTLA4-FasL effect.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

According to at least some embodiments of the present invention, thereis provided methods of treatment of lymphoma and/or multiple myelomathrough the administration of chimeric proteins which are useful forboth blocking and signaling.

According to at least some embodiments, the chimeric protein is providedin a pharmaceutical composition, comprising the protein and apharmaceutically suitable carrier.

A pharmaceutical composition according to at least some embodiments ofthe present invention can be administered via one or more routes ofadministration using one or more of a variety of methods known in theart. As will be appreciated by the skilled artisan, the route and/ormode of administration will vary depending upon the desired results.Preferred routes of administration for therapeutic agents of theinvention include intravascular delivery (e.g. injection or infusion),intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous,spinal, oral, enteral, rectal, pulmonary (e.g. inhalation), nasal,topical (including transdermal, buccal and sublingual), intravesical,intravitreal, intraperitoneal, vaginal, brain delivery (e.g.intra-cerebroventricular, intra-cerebral, and convection enhanceddiffusion), CNS delivery (e.g. intrathecal, perispinal, andintra-spinal) or parenteral (including subcutaneous, intramuscular,intravenous and intradermal), transmucosal (e.g., sublingualadministration), administration or administration via an implant, orother parenteral routes of administration, for example by injection orinfusion, or other delivery routes and/or forms of administration knownin the art. The phrase “parenteral administration” as used herein meansmodes of administration other than enteral and topical administration,usually by injection, and includes, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural and intrasternal injection and infusion.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the chimeric protein may be coated in amaterial to protect the protein from the action of acids and othernatural conditions that may inactivate the protein.

A pharmaceutical composition according to at least some embodiments ofthe invention also may include a pharmaceutically acceptableanti-oxidant. Examples of pharmaceutically acceptable antioxidantsinclude: (1) water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Examplesof suitable aqueous and nonaqueous carriers that may be employed in thepharmaceutical compositions according to at least some embodiments ofthe invention include water, ethanol, polyols (such as glycerol,propylene glycol, polyethylene glycol, and the like), and suitablemixtures thereof, vegetable oils, such as olive oil, and injectableorganic esters, such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of coating materials, such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositionsaccording to at least some embodiments of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin. Sterile injectable solutionscan be prepared by incorporating the active compound in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, optionally from about0.1 percent to about 70 percent, optionally from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms according to at least some embodiments of theinvention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such an active compound for the treatment of sensitivity inindividuals.

For administration of the chimeric protein, the dosage ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host bodyweight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg bodyweight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weightor within the range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months.

Alternatively, the chimeric protein can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of thechimeric protein in the patient. The dosage and frequency ofadministration can vary depending on whether the treatment isprophylactic or therapeutic. In prophylactic applications, a relativelylow dosage is administered at relatively infrequent intervals over along period of time. Some patients continue to receive treatment for therest of their lives. In therapeutic applications, a relatively highdosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patient can be administered a prophylacticregime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions according to at least some embodiments of the presentinvention may be varied so as to obtain an amount of the activeingredient which is effective to achieve the desired therapeuticresponse for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions according to at least someembodiments of the present invention employed, or the ester, salt oramide thereof, the route of administration, the time of administration,the rate of excretion of the particular compound being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

A “therapeutically effective dosage” of a chimeric protein according toat least some embodiments of the invention preferably results in adecrease in severity of disease symptoms, an increase in frequency andduration of disease symptom-free periods, an increase in lifespan,disease remission, or a prevention of impairment or disability due tothe disease affliction. For example, for the treatment of MultipleMyeloma, lymphoma and/or leukemia, a “therapeutically effective dosage”optionally inhibits cell growth or tumor growth by at least about 20%,40%, 60%, 80% relative to untreated subjects. The ability of a compoundto inhibit tumor growth can be evaluated in an animal model systempredictive of efficacy in human tumors. Alternatively, this property ofa composition can be evaluated by examining the ability of the compoundto inhibit, such inhibition in vitro by assays known to the skilledpractitioner. Alternatively or additionally, the suitable amount ordosage may also optionally be at least partially selected according tothe administration of one or more additional treatments for multiplemyeloma, which may optionally and preferably have a synergistic effectand so which may optionally cause the dosage amount to be adjusted. Suchone or more additional treatments could easily be selected by one ofordinary skill in the art.

Alternatively or additionally, a “therapeutically effective dosage”preferably results in at least stable disease, preferably partialresponse, more preferably complete response, as assessed by the WHO orRECIST criteria for tumor response (Natl Cancer Inst 1999; 91:523-8 andCancer 1981; 47:207-14).

A therapeutically effective amount of a therapeutic compound candecrease tumor size, or otherwise ameliorate symptoms in a subject, orotherwise support partial or complete stable disease and/or partial orcomplete response as determined above. One of ordinary skill in the artwould be able to determine such amounts based on such factors as thesubject's size, the severity of the subject's symptoms, and theparticular composition or route of administration selected.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition according to at least some embodiments of the invention canbe administered with a needles hypodermic injection device, such as thedevices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335;5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-knownimplants and modules useful in the present invention include: U.S. Pat.No. 4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a controlled rate; U.S. Pat. No. 4,486,194,which discloses a therapeutic device for administering medicamentsthrough the skin; U.S. Pat. No. 4,447,233, which discloses a medicationinfusion pump for delivering medication at a precise infusion rate; U.S.Pat. No. 4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. These patents are incorporated herein byreference. Many other such implants, delivery systems, and modules areknown to those skilled in the art.

In certain embodiments, the chimeric protein according to at least someembodiments of the invention can be formulated to ensure properdistribution in vivo. For example, the blood-brain barrier (BBB)excludes many highly hydrophilic compounds. To ensure that thetherapeutic compounds according to at least some embodiments of theinvention cross the BBB (if desired), they can be formulated, forexample, in liposomes. For methods of manufacturing liposomes, see,e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomesmay comprise one or more moieties which are selectively transported intospecific cells or organs, thus enhance targeted drug delivery (see,e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplarytargeting moieties include folate or biotin (see, e.g., U.S. Pat. No.5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem.Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995)FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother.39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. JPhysiol. 1233:134); p 120 (Schreier et al. (1994) J. Biol. Chem.269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett.346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

Example 1—Efficacy of Chimeric Protein for Treatment of HematologicalMalignancies

Materials and Methods

Cells, Abs, Reagents, and Fusion Proteins

Daudi, Raji, and JY and 293 human kidney cell lines were originallyobtained from the American Type Culture Collection (ATCC) (Bethesda,Md., USA). Two Jurkat sublines (J-CD40L+ and J-CD40L−) were kindlyprovided by Dr. John Fayen (CWRU. Cleveland, Ohio, USA). All cell lineswere followed weekly to verify that they retained their originalappearance and growing rates, and remained free of contamination. Also,cells were repeatedly tested to verify continued expression of expectedsurface molecules. If any change was suspected, new lots were thawed.Medium was tested for mycoplasma contamination using a commercial PCRkit (Biological Industries. Israel).

FITC-conjugated fluorescent Ab specific for CD40L, CD80, CD86 and CD95,along with their matched FITC-conjugated IgG isotypes, were purchasedfrom PharMingen (San Diego, Calif., USA). Recombinant human CTLA-4⋅Ig(CTLA-4/Fc) and sFasL were purchased from R&D Systems (MN, USA) andAlexis Biochemicals (San Diego, Calif.), respectively. CD40-Fc fusionprotein was purchased from Calbiochem (Darmstadt, Germany). Anti-humanB7-1 & B7-2 (CD80 and 86) Ab were purchased from R&D Systems. ForWestern blot analysis, anti-β actin Ab and anti-mouse GAPDH Ab werepurchased from Sigma-Aldrich and Chemicon International, respectively.Anti-FLIPS/L and anti-Caspase 8 pAb were purchased from Santa CruzBiotechnology (Santa Cruz, Calif. USA) and MBL (Medical & BiologicalLaboratories Co, USA), respectively. Anti-Caspase 3 & 9 pAb werepurchased from Cell Signaling Technology (Danvers, Mass.).

Culture medium for all experiments was RPMI 1640 (Biological Industries,Israel), supplemented with 10% FBS (Invitrogen Life Technologies, CA), 2mM L-glutamine, and 100 U/ml penicillin/streptomycin (BiologicalIndustries).

A hexahistidine-tagged derivative of CTLA-4⋅FasL (his6CTLA-4⋅FasL), withthe tag appended to the amino terminus, and CD40⋅FasL were prepared aspreviously described in Huang J H and Tykocinski M L (CTLA-4-Fas ligandfunctions as a trans-signal converter protein in bridgingantigen-presenting cells and T cells. Int Immunol 2001, 13:529-539);Elhalel M D et al (CTLA-4⋅FasL induces alloantigen-specifichyporesponsiveness. J Immunol 2003, 170:5842-5850); andDranitzki-Elhalel M et al (CD40⋅FasL inhibits human T cells: evidencefor an auto-inhibitory loop-back mechanism. Int Immunol 2007,19:355-363), respectively.

Proliferation Assays

Jurkat, Daudi or Raji cells in exponential growth phase were washedtwice and re-suspended in medium at 1×106 cells/ml. 50 μl of cellsuspension was added to individual wells of round-bottom 96-well tissueculture plates. CTLA-4⋅FasL, CD40⋅FasL, sFasL, CTLA-4⋅Ig or CD40⋅Fc, orcombinations of the latter three were added at different concentrations.Total culture volume was 200 μl/well. In some experiments, anti-CD80 oranti-CD86 Ab were added 20 min prior to the addition of CTLA-4⋅FasL. Inother experiments. CTLA4⋅Ig in PBS was added to flat-bottom 96-wellplates and incubated for 1 h at 370 C and then overnight at 40 C, inorder to pre-coat the plates with CTLA4⋅Ig. The next day, plates werewashed 4 times with PBS, and 5×104 Raji cells were added to each welland incubated for 24 h. Cultures were then pulsed with 0.5 μCi of[3H]thymidine (PerkinElmer, Waltham, Mass., USA) and incubated at 37°C., 6% CO2 and 95% humidity for 18-24 hours. Cells were subsequentlyharvested onto glass fiber filters for scintillation counting. Allproliferation assays were performed in triplicate.

Flow Cytometry

Cells were washed twice with FACS buffer (0.5% BSA/0.02% sodium azide in1×PBS) and incubated on ice for 30-45 min with one of the following;FITCconjugated anti-CD40L Ab, anti-CD80 Ab, anti-CD86 Ab, anti-CD95 Ab,or their matching isotype controls, all purchased from PharMingen (SanDiego, Calif., USA). Flow cytometry was performed using a FACSCaliburflow cytometer (Becton Dickinson, San Jose, Calif., USA), and data wereanalyzed using CellQuest software (Becton Dickinson). A total of 1×105events were collected for each sample.

To track cells undergoing apoptosis, 1×106 Jurkat T cells or 1.5×106Raji or Daudi B cells were incubated in 24-well plates in a total volumeof 1 ml, in the presence or absence of one of the following:CTLA-4⋅FasL, CD40⋅FasL, sFasL, CTLA4⋅Ig or CD40⋅Fc or combinations ofthe latter three. After 4 h (T cells) or 16 h (B cells), cells werecollected and washed twice with cold FACS buffer (0.5% BSA/0.02% sodiumazide in 1×PBS). For detection of apoptosis and necrosis, cells wereco-stained with propidium iodide (PI) and annexin-VFITC using a kit(MBL, Medical & Biological Laboratories Co, USA), according to themanufacturer's protocol. Flow cytometry was performed using aFACSCalibur flow cytometer, and data were analyzed using CellQuestsoftware. A total of 1×105 events were collected for each sample. Wholecell lysates and Western blotting analysis

Jurkat, Raji, JY or Daudi cells in exponential growth phase were washedtwice, resuspended in medium at 5×106 cells/ml, and plated in 24-wellplates in a total volume of 1 ml. CTLA-4⋅FasL, CD40⋅FasL, sFasL,CTLA4⋅Ig or CD40-Fc, or combinations of the latter three were added atdifferent concentrations. After 90′, cells were collected, washed twicewith ice-cold PBS, and lysed in lysis buffer (0.5% Nonidet P-40, 50 mMTris-HCl (pH 8.0), 100 mM NaCl, 1 mM PMSF, 1 mM sodium orthovanadate, 10μg/ml leupeptin, and 10 μg/ml aprotinin) for 20-30 min on ice. Theprotein concentration of whole cell lysates was determined using theBio-Rad Protein Assay Kit (Bio-Rad, Richmond, Calif.), according to themanufacturer's protocol. Whole cell lysates were mixed with Laemmlisample buffer (Bio-Rad) at 1:1 ratio, heated for 10 min at 95° C., andequal amounts of protein were loaded onto 10% SDSPAGE.

Following electrophoresis, gels were blotted onto nitrocellulosemembranes (Schleicher & Schuell), blocked with 5% milk/PBS, and probedovernight with primary Ab. After extensive washing, blots were incubatedwith HRP-conjugated matching secondary Ab (Bio-Rad), and developed withenhanced chemiluminescent substrate (Sigma-Aldrich) before exposure toXray film. Films were scanned and quantified by ImageMaster VDS-CL(Amersham Pharmacia Biotech). All membranes were re-blotted with eitheranti-β actin mAb or anti-GAPDH mAb to verify that similar quantities ofprotein were loaded on the gel.

Results

Fusion Protein-Mediated Inhibition of Cell Proliferation is Dependent onthe Surface Expression of Cognate Receptors

As a first step, the functionality of the component parts of theCTLA-4⋅FasL and CD40⋅FasL fusion proteins was established. To this end,malignant B (Raji, JY and Daudi) and T (Jurkat) cell lines that differin the expression of counter-receptors for these fusion proteinelements—namely B7 (CD80 and CD86), CD40 ligand (CD40L) and Fas receptor(CD95)—were used. In the case of Jurkat, two sublines that differ intheir expression of CD40L (J-CD40L+ versus J-CD40L−) 26 were paired. Tostart, surface expression of the molecules on these various cell lineswas verified by immunofluorescence and flow cytometry (FIG. 1). Asexpected, the B and T cell lines were positive and negative for B7molecules, respectively, and Daudi cells expressed negligible levels ofFas receptor. The difference in CD40L expression between the J-CD40L+and J-CD40L− T cell sublines was also confirmed.

Next, it was determined whether the differences in cognate surfacereceptor expression correlate with the abilities of CTLA-4⋅FasL andCD40⋅FasL to inhibit the proliferation of the tumor lines (FIG. 2A).Specifically, the different cell lines were pulsed with [3H]thymidinefor 16-20 h in the presence or absence of CTLA-4⋅FasL, CD40⋅FasL,CTLA4⋅Ig, CD40-Fc, sFasL, or different combinations of the latter three.As expected, the proliferation of Daudi cells, which express minimal Fasreceptor, was not inhibited by either of the FasL-containing fusionproteins, CTLA-4⋅FasL and CD40⋅FasL, nor by sFasL (FIG. 2A, lowestpanel), highlighting the Fas-dependence of their inhibitory activity. Incontrast, these fusion proteins significantly inhibited proliferation ofthe other two malignant B cell lines, Raji (FIG. 2A) and JY (not shown),which do express the Fas death receptor. CTLA-4⋅FasL was substantiallymore potent than sFasL and CTLA4⋅Ig, alone or in combination, ininhibiting proliferation of the B7-expressing malignant B cells. Indeed,CTLA4⋅Ig had no effect on proliferation at concentrations as high as 100ng/ml.

Of note, CTLA-4⋅FasL was significantly more effective than CD40 FasLagainst the B cell lines (which are negative for CD40L). In fact, CD40FasL, at concentrations up to 30 ng/ml, had no inhibitory effect on theB cell lines.

To further explore the requirement for CD40L expression on target cellsfor CD40⋅FasL's activity, advantage was taken of the Jurkat T cellderivative lines that differ in CD40L expression (J-CD40L+ versusJ-CD40L−). Both of these T cell lines were inhibited by sFasL andFasL-containing fusion proteins to a greater extent than Raji B cells(FIG. 2A, upper two panels), with the JCD40L− line being the mostsensitive. Interestingly, as shown previously, only cells that expressCD40L (J-CD40L+) were affected by low concentrations of CD40⋅FasL,though at higher concentrations of CD40⋅FasL (e.g. >100 ng/ml), allFas-expressing cells were inhibited to some degree (not shown).

Given that T cells were highly sensitive to all forms of sFasL, it wasnot possible to use them as B7-negative controls for establishing therequirement for B7 surface expression in CTLA-4⋅FasL's activity.Consequently, an Ab blocking experiment was performed using B7-positiveB cells as targets. In particular, CTLA-4⋅FasL's inhibitory effect inthe absence or presence of antagonistic Ab against CD80 and CD86 (FIG.2B) was compared. Anti-CD80 blocking Ab (0.5 or 1 μg/ml) significantlyattenuated the inhibitory effect of CTLA-4⋅FasL (30 ng/ml). In contrast,anti-CD86 Ab, at the same concentrations, had no effect on CTLA-4⋅FasLinhibition of Raji cell proliferation. The addition of the two blockingAb in combination completely abolished CTLA-4⋅FasL's inhibitory effect.Taken together, these data indicate that CTLA-4⋅FasL and CD40⋅FasLexhibit maximal inhibitory potency when the target malignant lymphoidlines express cognate counter-receptors for both domains of each fusionprotein.

Fusion Protein-Mediated Induction of Apoptosis is Dependent on theSurface Expression of Cognate Receptors

Previous studies have established that both CTLA-4⋅FasL and CD40⋅FasLinduce their inhibitory effect via Fas-mediated apoptosis. It was thenconsidered whether apoptosis induction by these fusion proteinsparallels proliferative inhibition in being more efficient when targetcells co-express surface molecules that can bind both ends of therespective proteins. To this end, the same set of malignant cell lineswere incubated for 4 (T cells) or 16 (B cells) hours in the presence orabsence of CTLA-4⋅FasL, CD40⋅FasL, CTLA4-Ig, CD40-Fc, sFasL, ordifferent combinations of latter three. At the end of the treatmentperiod, cellular apoptosis and necrosis were assessed using Annexine/PIstaining and flow cytometry. Again as expected, FasLcontaining fusionproteins or sFasL had no effect on Daudi B cells lacking Fas receptor,and neither CTLA-4⋅Ig or CD40-Fc induced apoptosis in the Daudi cells(FIG. 3A, B). By contrast, and as was the case in the proliferationassay, CTLA-4⋅FasL significantly increased the percentage of apoptoticor necrotic Raji cells at 16 h (FIG. 3A, B). Again, more apoptosis wasdetected when Raji cells were incubated with CTLA-4⋅FasL, as opposed tosFasL, CTLA-4-Ig, or a combination of the latter two. No significantapoptosis of Raji cells was induced by CD40⋅FasL (FIG. 3A, C).

When Jurkat T cells (either J-CD40L+ or J-CD40L−) were incubated withCTLA-4⋅FasL, significant apoptosis ensued, though J-CD40L+ were lessaffected than J-CD40L− (FIG. 3A, C). The same difference insusceptibility to apoptosis induction between these paired cells lineswas evident when sFasL or the anti-Fas Ab CH11 (not shown) was used.However, when the Jurkat cell lines were incubated in the presence ofCD40⋅FasL, significant apoptosis could be detected only in J-CD40L+cells, despite the fact that sFasL was more potent in inducing apoptosisin J-CD40L− cells (FIG. 3A, C). This set of experiments highlights thefunctional importance of both ends of each of the respective fusionproteins for efficient apoptosis induction.

Both Pro-Apoptotic and Anti-Apoptotic Pathways are Affected byCTLA-4⋅FasL and CD40⋅FasL

The possibility that the apoptosis-inducing activity of CTLA-4⋅FasL andCD40 Fas may not be solely reliant on triggering of the Fas deathreceptor was then considered. To test this hypothesis, the T and B celllines were incubated for 90-180 min with CTLA-4⋅FasL, CD40⋅FasL, sFasL,CTLA-4⋅Ig, CD40 or combinations of the latter three. At the end of theincubation period, whole cell lysates were evaluated by immunoblottingfor expression of the anti-apoptotic protein cFLIP, caspase 8 (as amarker of the extrinsic pathway), caspase 9 (as a marker of theintrinsic, mitochondrial pathway), and caspase 3. Of note, both thepro-caspase forms (not shown) and the cleaved, active caspase forms wereexamined. A representative experiment is shown in FIG. 4A, and thesummary of 3 independent experiments performed with the different celllines is shown in FIGS. 4 B-D.

The expression of the different caspases in the Daudi B cell line (withnegligible Fas receptor) did not change after incubation with theFasLcontaining proteins (FIG. 4A). Furthermore, no change was found whenDaudi cells were incubated with CTLA-4⋅Ig, CD40-Fc or a combination ofthe latter with sFasL (FIG. 4A). In contrast, B cell lines co-expressingB7 molecules and Fas receptors yielded an entirely different picture.After 90 or 180 minutes of incubation in the presence of CTLA-4⋅FasL,cFLIPL expression was abrogated, and a clear increase in the activated(cleaved) forms of two of the caspases (9 and 3) was observed. Sincecaspase 8 is found mainly in its active, cleaved form in these cells,even when Fas receptor is not triggered and cFLIP base levels are verylow, another malignant B cells line, JY, also co-expressing B7 moleculesand Fas receptor, was considered.

When incubated with CTLA-4⋅FasL, there was a clear decrease in cFLIPabundance and a significant increase in the activated forms of caspases3, 9, and 8 (FIGS. 5A-C). In contrast, CD40⋅FasL, sFasL, CTLA-4⋅Ig, orCD40-Fc had no effect on cFLIP expression or caspase activation ineither of the malignant B cell lines (FIGS. 4 and 5).

The next focus was on CD40 FasL with an appropriate cell target,J-CD40L+. When the J-CD40L+ and J-CD40L− T cell lines were incubatedwith CTLA-4⋅FasL or CD40⋅FasL, the pattern expression of apoptotic andanti-apoptotic protein expression was distinctly different from thatseen with the B cell lines (FIG. 4A, upper two panels; FIG. 4C). In bothFas-expressing Jurkat cell lines, CTLA-4⋅FasL and CD40⋅FasL eachactivated the caspase cascade, showing increased abundance of theactivated forms of caspase 9 and 3.

Caspase 8 appears in its activated form in these cell lines. No changein cFLIP abundance was noted. However, J-CD40L+ cells, though lesssensitive to CTLA-4⋅FasL than J-CD40L− (as one reflected in the lesserincrease in the activated forms of caspase 9 and 3), responded toCD40⋅FasL treatment with a significant decrease in cFLIP abundance, andadditional activation of caspases 8, 3 and 9. CD40⋅FasL was more potentin doing so than CD40-Fc, sFasL or a combination of the two. Takentogether, these experiments establish that CTLA-4⋅FasL and CD40⋅FasLeach has dual and reinforcing effects on both apoptotic (caspase) andanti-apoptotic (cFLIP) pathways.

CTLA-4⋅Ig inhibits proliferation of a B cell line and decreases cFLIPlevels. One possible explanation for the special properties of thefusion proteins, with unique impact on anti-apoptotic signaling, isback-signaling through the non-Fas surface counter-receptors for thefusion protein components. Indeed back-signaling through the‘costimulatory ligands’ B7 (on antigen-presenting cells) and CD40L (on Tcells) has been documented by others. It was therefore consideredwhether the exceptional potency of the CTLA-4⋅FasL and CD40⋅FasL fusionproteins might be explained, at least in part, by backsignaling throughB7 and CD40L, respectively, occurring simultaneously with Fas receptortriggering. For that purpose, Raji B cells were treated with CTLA-4⋅FasLfor 24 h, in the presence or absence of plate-bound CTLA-4⋅Ig, andproliferation was assessed. As shown in FIG. 6A, plate-bound CTLA-4⋅Igalone significantly inhibited Raji cell proliferation.CTLA-4⋅FasL-mediated inhibition of Raji cell proliferation wasdiminished in the presence of plate bound CTLA-4⋅Ig, suggestinginterference with CTLA-4⋅FasL binding to B7 (FIG. 6B). Significantly,plate-bound CTLA-4⋅Ig, like CTLA-4⋅FasL, reduced cFLIP expression inRaji cells and increased the expression of caspases 9 and 3 in them(FIG. 6C). These findings suggest that CTLA-4⋅Ig can deliver aninhibitory back-signal to malignant B cells and reduce anti-apoptoticcFLIP in them.

It was considered whether CD40⋅FasL can mediate similar back-signalingthrough CD40L. In order to test this possibility, J-CD40L+ cells wereincubated with CD40-Fc. CD40-Fc increased J-CD40L+ proliferation by ˜20%(not shown). However, no effect on the expression of the apoptotic andantiapoptotic proteins was observed when CD40-Fc was used atconcentrations up to 1200 ng/ml (data not shown).

Discussion

In this study, the unique functional properties of two fusion proteins,CTLA-4⋅FasL and CD40⋅FasL, as inducers of apoptosis in malignantlymphoid cell lines have been considered, focusing on relevantintracellular signaling cascades. Without wishing to be limited by asingle hypothesis or a closed list, these findings include: 1)CTLA-4⋅FasL and CD40⋅FasL induce death in Fas receptor-expressingmalignant lymphoid lines of both B and T lineages; 2) CTLA-4⋅FasLinduces apoptosis in B7-expressing B cell lines more potently than doesCD40⋅FasL; 3) CD40⋅FasL induces apoptosis in CD40L-expressing J-CD40L+ Tcells in a CD40L-dependent fashion; 4) CTLA-4⋅FasL lowers cFLIPexpression and activates the caspase cascade in cells co-expressing bothB7 and Fas receptor at their surfaces; 5) CD40 FasL lowers cFLIPexpression and activates the caspase cascade in cells co-expressing bothCD40L and Fas receptor, and 6) CTLA-4⋅FasL and CD40⋅FasL are each moreeffective in inducing apoptosis than either of their component parts,alone or in combination.

Taken together these findings affirm the higher order functionality ofthese unique fusion proteins, and suggest that they may have specialadvantages for inducing apoptosis in malignant lymphoid cells, bycoordinately affecting apoptotic and anti-apoptotic pathways. One of themechanisms enabling malignant cells to escape apoptosis, and therebyremain refractory to treatment, is up-regulation of anti-apoptoticproteins. FLIP, expressed either intrinsically (as cFLIP) orextrinsically (as vFLIP), has been implicated as a key anti-apoptoticprotein in several malignant lymphomas. It has been previously shownthat CTLA-4⋅FasL prevents the up-regulation of cFLIP expression thatnormally accompanies T cell activation. By abrogating up-regulation ofthis anti-apoptotic protein, CTLA-4⋅FasL is able to induce apoptosis inactivated T cells at an earlier phase than does sFasL. The present studyextends this functional feature of CTLA-4⋅FasL to transformed cells.

For most of the cell lines studied here (that is, J-CD40L+, J-CD40L− andJY cells), reduction in cFLIP expression was correlated with activationof caspases 3, 8, and 9. Caspase 8 activation is dependent on itsbinding to the death complex FADD. This binding leads to activation ofcaspase 3, and Bid, that in turn results in cytochrome c and caspase 9activation. Activation of caspase 9 then leads to more caspase 3activation and effective apoptosis. The ability of cFLIP proteins toinhibit caspase 8 activation, by competing with caspase 8 for binding tothe death complex FADD, is well-established.

There are contradictory data in the literature concerning the role ofthe two splice variants of cFLIP:cFLIP short (cFLIPs) and cFLIP long(cFLIPL). cFLIPL's role in the system appears to be more complicated,with data indicating that high levels of expression lead to apoptosis,whereas moderate levels result in the opposite, that is, inhibition ofFas-mediated apoptosis in vitro and in vivo. However, data are clearerin the case of cFLIPL under-expression, in that selective silencing ofcFLIPL mRNA augments caspase 8 recruitment, activation, processing andrelease from the death complex and hence enhanced apoptosis. Only thecFLIPL splice variant was detected in the Raji B cell line, andCTLA-4⋅FasL, able to bind to both B7-1 (CD80) and Fas receptor (CD95) onthese cells, decreased expression of this cFLIP isoform. Significantly,despite the constitutive expression of the activated form of caspase 8in these Raji cells. CTLA-4⋅FasL-driven cFLIPL reduction was correlatedwith activation of caspases 9 and 3 and effective induction ofapoptosis. Without wishing to be limited by a single hypothesis,reduction of cFLIP by CTLA-4⋅FasL and CD40⋅FasL may lead to bothpro-apoptotic and anti-proliferative effects in transformed lymphoidcells.

By chimerizing FasL to CTLA-4 and CD40, one creates intriguing molecularbridging possibilities. Transformed B cells introduce a new scenariowherein the same fusion protein can potentially bridge thecounter-receptors being co-expressed on the same cell. Intercellular andintracellular bridging by both CTLA-4⋅FasL and CD40⋅FasL are certainlynot mutually exclusive, and one can envision both taking place in thecontext of local tumor growth.

The cis loop-back auto-signaling mechanism may lead to more effectiveinhibition on more than one basis. First, the fusion proteins serve totether FasL to membranes, via either CTLA-4:B7 binding on B cells orCD40:CD40L binding on T cells. The potency of surface-anchored FasL hasbeen clearly established in the context of exogenously-introducing itonto APC surfaces to generate deletional APC 56-59. One would thusexpect fusion protein-tethered FasL to be highly functional in anauto-signaling mode as well. Second, both CTLA-4⋅FasL and CD40⋅FasL havethe potential to act as dual-signaling agents by triggering neighboringcounter-receptors on the same cell surface.

Back-signaling through the B7 molecules CD80 and CD86 has been describedfor B cells 23, leading to proliferative inhibition of B cells in thecase of CD80 and the opposite for CD86. Since CTLA-4 has higher avidityfor CD80, one might expect the CD80-related effect to dominate forCTLA-4⋅FasL. Data here showing that CTLA-4⋅Ig inhibits proliferation ofCD80-expressing Raji cells is consistent with this back-signalingfunction.

Back-signaling has also been demonstrated for CD40L on T cells, and thuscould also contribute to CD40⋅FasL's observed efficacy. In the case ofCD40⋅FasL, but not CD40-Fc, the CD40 moiety is being presented in acellbound mode (via FasL:Fas anchoring), and with the likelihood ofbeing in a trimer or two-trimer configuration (versus the presumedCD40-Fc dimer).

Indeed, it has been shown that CD40-Fc has lower affinity for CD40L thando oligomers of CD40, and this higher affinity is accompanied bystronger biological activity. Taken together, but without wishing to belimited by a single hypothesis, these various data suggest that not onlycan each of CTLA-4⋅FasL and CD40⋅FasL deliver dual signals on the samecell, but in both instances, the Fas signaling can be potentiated bybacksignaling through the other counter-receptor (B7-1 or CD40L).

Of note, CTLA-4⋅FasL was found to be a potent inducer of apoptosis inJurkat T cells, even though these cells are negative for B7 proteins.This likely reflects their overall higher susceptibility to solubleFasL-mediated apoptosis, as evidenced by their high sensitivity(especially the J-CD40L− subline) to sFasL and agonistic anti-Fas Ab(CH11; data not shown).

The present study highlights the functional richness of appropriatelyconfigured fusion proteins. The CTLA-4⋅FasL and CD40⋅FasL fusionproteins modulate both normal and transformed lymphoid cells, serve tobridge molecules intercellularly and intracellularly, set up artificialcis loop-back autosignaling loops, and deliver dual-signals that arereinforcing. Both of these proteins can down-modulate anti-apoptoticcFLIP isoforms, an effect that serves to potentiate the FasLpro-apoptotic signal that these same proteins deliver. Thus, one canleverage fusion proteins to simultaneously deliver a death signal andsensitize the cells to it.

Example 2—Clinical Trial of a Chimeric Protein for Treatment of aHematological Malignancy

A phase I trial of a chimeric protein (therapeutic agent) as describedherein to subjects with a hematological malignancy selected from thegroup consisting of lymphoma, multiple myeloma and a leukemia as recitedherein, may be designed to evaluate both effect on disease progressionand possible toxicity. Subjects with a suspected hematologicalmalignancy may be enrolled after positive diagnosis of the hematologicalmalignancy as is well known in the art.

Treatment may be provided as a single therapeutic or in combination withaccepted treatments. For example, treatment strategies for multiplemyeloma are reviewed in Rajkumar et al., 2002, Mayo Clin. Proc. 77:814,hereby incorporated by reference in its entirety). Recognition of acuteor unusual progression of the disease may halt administration oftherapeutic agent.

Initial subjects receive a suitable dosage of therapeutic agent,administered for example as a 1 hour intravenous infusion or asappropriate, alone or in combination with one or more other known agentsas could be selected by one of ordinary skill in the art. Given adequatetolerance, the dose will be increased stepwise in subsequent subjects.Additionally, the method for administration may be changed to bolusinjection.

Toxicity of therapeutic agent is evaluated in subjects according to theWorld Health Organization Toxicity Criteria: blood pressure, temperatureand heart rate are monitored every 10 minutes during infusion, thenevery hour for 3 hours and finally every 3 hours for 24 hours.Hematologic, renal and liver function tests are conducted every otherday for one week and on day 15, 30, 60 and 120 post injection.

Serum and/or tissue samples are obtained once a week for two months sothat the effects of the therapeutic agent may be determined by methodsknown in the art, e.g., change serum concentration of M protein, changein hemoglobin value, presence/regression of lytic bone lesions etc.Pathologic studies will assess treatment effect on tissue damageassociated with the hematological malignancy.

Example 3—Effect on Tumor Cell Lines

Materials and Methods.

Unless otherwise stated, all chemicals were obtained from SIGMA(Israel). DMEM medium, FBS, PBS, Trypsin-EDTA, penicillin, streptomycinand L-Glutamine were obtained from Biological Industries (Beit Haemek,Israel).

Cell Lines.

Raji (EBV transformed B cell lymphoma line), JY (EBV transformed B celllymphoma line), Daudi (EBV transformed B cell lymphoma line) RPMI 8226(human multiple myeloma cell line), HL60 (Human promyelocytic leukemiacells) were purchased from ATCC (USA). SK-HEP-1 (HTB-52; liveradenocarcinoma cell line) was purchased from ATCC (USA). HepG2. Huh7hepatocellular carcinoma cell lines, originally from the ATCC, werekindly provided by the Hepatology Unit, Hadassah Hebrew UniversityMedical Center in Jerusalem, Israel. Unless otherwise stated, all celllines were grown in 10% FBS DMEM supplemented with 10 U/ml penicillin,0.1 mg/ml streptomycin and 292 μg/ml L-Glutamine. All cell lines werecultured at 37° C. in 6% CO2 and cultures were tested periodically formycoplasma contamination using EZ-PCR mycoplasma test kit (BiologicalIndustries).

Activity Assay.

To assess KAHR-102 (CTLA4-FasL) activity, 0.2×106 cells/ml were seededas triplicates in 96-well plates (NUNC, Roskilde, Denmark) and culturedwith or without varying concentrations of the histidine tagged versionof KAHR-102 (his6CTLA4-FasL), Cells were incubated with the indicatedprotein concentration for 24 h at 37° C. in 6% CO2, and cell viabilitywas evaluated using MTS assay (Promega, Madison, USA).

Flow Cytometry.

To assess apoptosis, 0.2×106 cells/ml were seeded as duplicates in24-well plates (NUNC) and incubated with or without varyingconcentrations of his6CTLA4-FasL (KAHR-102), soluble FasL, CTLA4-Fc orthe latter in combination for 24 h. Cells were then harvested, andapoptotic cells were detected by flow cytometric analysis, using theAnnexinV/PI MEBCYTO Apoptosis Kit (MBL, Nagoya Japan), according to themanufacturer's protocol. 20,000 events per sample were counted using aFACSCalibur flow cytometer (Becton Dickinson, San Jose, Calif., USA),and data were analyzed using CellQuest software (Becton Dickinson).

To examine expression of Fas receptor and the B7 molecules, CD80 andCD86, in the different cell lines, cells were retrieved, washed instaining buffer (PBS containing 1% BSA and 0.1% sodium azide), andstained with phycoerythrin-labeled mAb with specificity for the abovementioned molecules or the relevant control Abs, at concentrationsrecommended by the manufacturer (eBioscience). Flow cytometry wasperformed using a FACSCalibur flow cytometer (Becton Dickinson), anddata were analyzed using CellQuest software (Becton Dickinson). A totalof 20,000 events were collected for each sample.

Statistical Analysis.

Data are presented as means±SD. Statistical comparison of means wasperformed by a two-tailed unpaired Student's t test. Differences with ap<0.05 were considered statistically significant.

Establishment of Tumor Xenografts.

Athymic BALB/c nu/nu nude male mice (Harlan. Israel), 4-6 weeks of age,maintained under defined flora conditions at the Hebrew UniversityPathogen-Free Animal Facility will be used. All experiments wereapproved by the Animal Care Committee of the Hebrew University. Raji, JYor RPMI 8226 grown to 80% confluence, harvested, washed with PBS, andinjected subcutaneously (2×107/mouse) into the right flanks of mice orintraperitoneal. Once palpable, tumors will be measured for their widthsand lengths using a micro-caliper for the subcutaneous tumors, and tumorvolumes were calculated (w2× length/2), or, for the intraperitonealtumors, abdominal diameter will be measured, and mice will be weighted.Mice will treated daily with subcutaneous injections of his6CTLA4-FasL(KAHR-102), (200 μg) for 8 days. If tumor will re-grow or re-appear,another treatment will follow in two weeks. Control groups will beinjected with similar volumes of the his6CTLA4-FasL (KAHR-102) dilutionbuffer. Tumor volumes will monitored for approximately a 3 month, oruntil tumor size exceeded the threshold requiring sacrifice of theanimal. At the end of experiments, mice will be sacrificed, and tumorswill be harvested, measured and weighed, and analyzed.

Results

CTLA4-FasL's cytotoxic activity was evaluated against different lymphomacells. As shown in FIGS. 7 and 8, CTLA4-FasL induces death of both Rajiand JY, both of which are B-cell lymphatic cancer cell lines, in adose-dependent fashion. Of note, significant cell death was detected ata concentration as low as 0.1 ng/ml, corresponding to an EC50 of 0.4nmol/l. Cell death was detected at 24 h, and was more robust at 48 h,with almost no live cells detectable at 3 ng/ml CTLA4-FasL. Importantly,primary B cells taken from healthy volunteers were resistant toCTLA4-FasL's toxic activity (not shown).

The human myeloma cell line, RPMI 8226, was then tested and as seen inFIG. 9 found that though their sensitivity to CTLA4-FasL is lower thanthat of the B cell lymphoma cell line, they do respond to higherconcentrations of CTLA4-FasL. In contrast, promyelocytic leukemia cellswere resistant to CTLA4-FasL's cytotoxic effect (FIG. 10).

The difference in susceptibility to CTLA4-FasL's action can be explainedby the different expression of the surface molecules capable of bindingthe fusion protein. The expression of CD80 and CD86, which bind CTAL4,and the expression of CD95 (Fas receptor, which binds FasL) were thentested on the four cell lines. As can be seen in FIGS. 11-14, the twohighly sensitive cell lines, namely Raji and JY, express all threesurface molecules. The RPMI 8226, human multiple myeloma cell line, thatexhibited intermediate sensitivity to CTLA4-FasL express both the Fasreceptor and CD86, however in lower levels than the B cell lines, and donot express CD80 at all. The promyelocitic leukemia cells, that are notaffected by CTLA4-FasL show low level of expression of CD86, and verylow levels of Fas receptor.

The analysis was extended to other cancer cell lines, namely, SK-Hep1hepatoma cells. As can be seen in FIG. 15, CTLA4-FasL exhibited acytotoxic effect against this tumor line, albeit with somewhat differentkinetics; CTLA4-FasL was by far more potent than CTAL4-Fc, soluble FasLor the combination of the latter. Using this cell line, the effect ofinhibition of apoptosis mediated by caspases on CTLA4-FasL's effect wastested. As shown in FIG. 16, the pan-caspase inhibitor, zVAD completelyabolished CTLA4-FasL effect, indicating that its cytotoxic effect isapoptosis based.

Whereas particular embodiments of this invention have been describedabove for purposes of illustration, it will be evident to those skilledin the art that numerous variations of the details of the presentinvention may be made without departing from the invention as defined inthe appended claims.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention.

The invention claimed is:
 1. A method of treating multiple myeloma,benign plasma cell disorder or premalignant forms of multiple myeloma ina subject, comprising administering to the subject a CD40-FasL chimericprotein, wherein cells of said multiple myeloma, said benign plasma celldisorder or said premalignant forms of multiple myeloma express CD40L.2. The method of claim 1, wherein said multiple myeloma is selected fromthe group consisting of multiple myeloma cancers which produce lightchains of kappa-type and/or light chains of lambda-type; aggressivemultiple myeloma; smoldering multiple myeloma (SMM), indolent multiplemyeloma, and primary amyloidosis.
 3. The method of claim 2, wherein saidaggressive multiple myeloma is primary plasma cell leukemia (PCL). 4.The method of claim 1, wherein said benign plasma cell disorder isselected from the group consisting of MGUS (monoclonal gammopathy ofundetermined significance) and/or Waldenstrom's macroglobulinemia (WM,also known as lymphoplasmacytic lymphoma) which may proceed to multiplemyeloma.
 5. The method of claim 4, to halt the progression of the benignplasma cell disorder to a malignant form of multiple myeloma.
 6. Themethod of claim 1, wherein said chimeric protein is administered as apharmaceutical composition comprising said chimeric protein and apharmaceutical carrier, adapted for treatment of said multiple myeloma,said benign plasma cell disorder or said premalignant forms of multiplemyeloma.
 7. The method of claim 6, wherein said multiple myeloma isselected from the group consisting of multiple myeloma cancers whichproduce light chains of kappa-type and/or light chains of lambda-type;aggressive multiple myeloma; smoldering multiple myeloma (SMM), indolentmultiple myeloma, and primary amyloidosis.
 8. The method of claim 7,wherein said multiple myeloma is primary plasma cell leukemia (PCL). 9.The method of claim 6, wherein said benign plasma cell disorder isselected from the group consisting of MGUS (monoclonal gammopathy ofundetermined significance) and/or Waldenstrom's macroglobulinemia (WM,also known as lymphoplasmacytic lymphoma) which may proceed to multiplemyeloma.
 10. The method of claim 9, to halt the progression of thebenign plasma cell disorder to a malignant form of multiple myeloma. 11.A method of treating multiple myeloma, benign plasma cell disorder orpremalignant forms of multiple myeloma in a subject, comprisingadministering to the subject a CTLA4-FasL chimeric protein.
 12. Themethod of claim 11, wherein cells of said multiple myeloma, said benignplasma cell disorder or said premalignant forms of multiple myelomaexpress B7.
 13. The method of claim 11, wherein said multiple myeloma isselected from the group consisting of multiple myeloma cancers whichproduce light chains of kappa-type and/or light chains of lambda-type;aggressive multiple myeloma; smoldering multiple myeloma (SMM), indolentmultiple myeloma, and primary amyloidosis.
 14. The method of claim 13,wherein said aggressive multiple myeloma is primary plasma cell leukemia(PCL).
 15. The method of claim 11, wherein said benign plasma celldisorder is selected from the group consisting of MGUS (monoclonalgammopathy of undetermined significance) and/or Waldenstrom'smacroglobulinemia (WM, also known as lymphoplasmacytic lymphoma) whichmay proceed to multiple myeloma.
 16. The method of claim 15, to halt theprogression of the benign plasma cell disorder to a malignant form ofmultiple myeloma.
 17. The method of claim 11, wherein said chimericprotein is administered as a pharmaceutical composition comprising saidchimeric protein and a pharmaceutical carrier, adapted for treatment ofsaid multiple myeloma, said benign plasma cell disorder or saidpremalignant forms of multiple myeloma.
 18. The method of claim 17,wherein cells of said multiple myeloma, said benign plasma cell disorderor said premalignant forms of multiple myeloma express B7.
 19. Themethod of claim 17, wherein said multiple myeloma is selected from thegroup consisting of multiple myeloma cancers which produce light chainsof kappa-type and/or light chains of lambda-type; aggressive multiplemyeloma; smoldering multiple myeloma (SMM), indolent multiple myeloma,and primary amyloidosis.
 20. The method of claim 19, wherein saidmultiple myeloma is primary plasma cell leukemia (PCL).
 21. The methodof claim 11, wherein said benign plasma cell disorder is selected fromthe group consisting of MGUS (monoclonal gammopathy of undeterminedsignificance) and/or Waldenstrom's macroglobulinemia (WM, also known aslymphoplasmacytic lymphoma) which may proceed to multiple myeloma. 22.The method of claim 21, to halt the progression of the benign plasmacell disorder to a malignant form of multiple myeloma.